Autoradiography enhancer and method of use

ABSTRACT

Disclosed is an improved autoradiography enhancer composition and method of use. The enhancer composition uses an acid anhydride as a dehydration agent.

This is a division of U.S. application Ser. No. 07/124,827, filed Nov.24, 1987, now U.S. Pat. No. 4,871,480 issued Oct. 13, 1989.

BACKGROUND OF THE INVENTION

The present invention relates to autoradiography compositions andmethods. More particularly, the invention relates to methods andcompositions useful in autofluorography, specifically compositions whichenhance the detection of radiolabelled substances.

Autoradiography is the process by which an image is generated in aradiation sensitive material, e.g., a photographic emulsion, whenexposed to radioactivity. This technique is particularly useful foridentification of biological substances such as proteins, amino acids,or nucleic acids. Autoradiographic techniques involve radiolabelling theprotein or nucleic acid with a radioactive isotope, and then determiningthe location of the radiolabelled molecule by allowing the radioactiveemissions to expose a photoemulsion on the photographic plate orspecimen coated with a photographic emulsion. Commonly used radioactiveisotopes used for radiolabelling are ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I and ³² P.The radiolabelled material may be in a chromatograph, an electrophoresisgel, or a tissue specimen.

A major problem in autoradiography is that the low energy and/orradiation level emitted by many of these isotopes when incorporated intoa protein or nucleic acid is insufficient to provide clear, rapidexposure of the photographic emulsion. Scintillators or fluors are usedin order to achieve this end. Fluors are molecules that absorb theradiation emitted by the radioactive isotopes and emit light. Theemitted light is more efficient at exposing the photographic emulsionthan the emissions from the radioactive decay, leading to quicker andmore accurate results.

In some circumstances, a scintillation solvent and/or a secondary fluoris incorporated into the solution in order to improve the efficiency offluorescence. Scintillation solvents are chemicals which assist in thetransfer of the emitted electron radiation from the radioactive isotopesto the fluors, improving the energy transfer and system efficiency. Thesecondary fluors absorb energy from the primary fluor and emit light ata different wavelength which may improve exposure of the photographicemulsion. The use of the fluor or scintillator system in thisimage-creating process is called autofluorography.

Autofluorography is useful with a wide variety of separation media andtechniques. These include thin layer chromatography, paperchromatography, gel electrophoresis, and column chromatography. Theradioactive material may be absorbed or adsorbed to the separationmedium. Media which are useful include silica gel, alumina, cellulose,polyamide, polyacrylamide, cross-linked dextran, agarose, andnitrocellulose. These media are normally supported by a plate or otherstructure.

Despite its broad applicability, there are substantial problems with theuse of autofluorography which make it a difficult method to apply. Forexample, in thin layer chromatography, where the radioactive emittersare at or near the surface of the chromatograph, they interact with thefluor in the surface layer. However, it is difficult to obtain a smooth,even distribution of the fluor even when using a spray technique forapplication. Uneven application can cause nonreproducible results.Additionally, since the fluor is normally dissolved in a carriersolvent, the radioactive material may migrate with the solvent, causingmovement of bands which also can lead to inaccurate results. Further,small crystals of the scintillator which form during the processing mayfall off or be moved during handling.

At the other extreme of media thickness, gel electrophoresis and otherrelated media separation techniques use much thicker media, typicallygreater than 0.1 mm. These thicker media lead to different problemsbecause the material to be detected is normally not located on thesurface area but rather is disbursed throughout the media. Since the lowenergy radiation of the incorporated radioactive isotopes is greatlyattenuated by distance, a surface treatment with the fluor isinsufficient to visualize a majority of the emitted radiation, leadingto inaccuracies. In order to counteract this, the fluor, either alone orin conjunction with an intermediate scintillation solvent and/orsecondary fluor, is placed in intimate contact with the radioactiveisotope to amplify the signal prior to attenuation. To achieve thiscontact, it is necessary to uniformly transfer the fluor systemthroughout the media which insures that there are no differences inresults caused by incomplete adsorption of emitted radiation. Generally,this uniform transfer is accomplished by soaking the separation mediumin a bath containing the fluor dissolved in a suitable solvent.

There are a number of different scintillation compositions and methodswhich have been used for enhanced autoradiography to identify materialsincorporated into separation media. One system is described by Bonner atal., Eur. J. Biochem. 46(1):83-88 (1974). In this method, theradiolabelled protein is separated by electrophoresis using an aqueouspolyacrylamide gel. Separation is followed by soaking the gel in about20 times its volume of dimethylsufoxide (DMSO) for 30 minutes, and thenimmersed a second time for 30 minutes in fresh DMSO to displace all thewater from the gel. The gel is then soaked in a 20% (w/w) solution of2,5-diphenyloxazole (PPO) in DMSO to impregnate the gel with thescintillator PPO. The fluor is precipitated in the gel by immersing inwater and the gel is dried and exposed to the photographic film.

The Bonner technique has a number of disadvantages, several of which arediscussed in the appendix of the Laskey and Mills article in Eur. J.Biochem., 56:335-341 (1975). For example, if agarose gels are used inplace of acrylamide, the agarose will dissolve in DMSO. This iscounteracted by the substitution of methanol for the DMSO but thissubstitution is only effective for gels having less than 2%polyacrylamide since gels having higher concentrations of polyacrylamideshrink severely when contacted with methanol. Another disadvantage isthe ability of DMSO to penetrate through the skin of anyone handling it.DMSO can carry the dissolved material, some of which may be radioactiveand otherwise potentially toxic, with it through the skin. DMSO alsoimparts a garlic smell to the breath. Other disadvantages reportedinclude the length of time gels must be soaked in the DMSO-fluorsolution to obtain complete impregnation, the requirement for highconcentrations of PPO, and the time-consuming and waste-generatingsoakings in DMSO to dehydrate the gel.

Because of these problems, many other methods for incorporating fluorsinto separation media appear in the literature. Bonner and Stedman,Anal. Biochem., 89:247-256 (1978), describe methods suitable for thinlayer chromatography. These methods use scintillation solvents toincrease the absorption ability of 2,5-diphenyloxazole. Although helpfulfor thin layer chromatography, these methods have serious drawbacks whenapplied to gel electrophoresis because the gels used generally containgreater than 80%, more often greater than 90%, water. The fluors andsolvents used in the Bonner and Stedman methods are not water soluble orwater miscible to any appreciable extent so they cannot be used forefficient and uniform transport of the fluors to the interior of thegel. In fact, the fluors tend to precipitate on the surface of the gel.In addition, the suggested organic solvents cause drastic shrinkage ofthe gel which prevents fluor impregnation and lead to distortion of thegel. In fact, aqueous polyacrylamide or agarose gels cannot beimpregnated with 2,5-diphenyloxazole or 2-methylnaphthalene while in thehydrated state using these methods.

U.S. Pat. No. 4,293,436, issued on an application of Fost, describes adifferent autofluorographic technique which does not require adehydration step. The aqueous separation medium is impregnated with acombination of a water-soluble or water-miscible lower alkyl carboxylicacid and an alcohol as a swelling inhibitor in which a scintillatorfluor has been dissolved. The fluor is precipitated within the gel byaqueous soaking. However, this procedure also has several disadvantages.Proteins must first be fixed in a separate step. Further, there islimited or no reusability of the excess product used for impregnation,which increases costs and generates hazardous wastes. In addition, thecombination of carboxylic acid and alcoholic swelling inhibitor isunstable and gradually forms a stable ester. This decreases thesuitability of the product for its intended use and limits shelf life.

Another autofluorographic system is described in U.S. Pat. No.4,522,742, issued on an application of Lee et al. In this technique, theseparation medium is impregnated by an aqueous autofluorographicenhancer containing water soluble fluors. This water-based system doesnot, however, work effectively in very thin gels (<1.0 mm) or gels withless than 5% acrylamide or agarose. No dehydration step is needed sinceunlike the systems based on organic solvent impregnation and waterprecipitation, this aqueous system transports fluor into the gel withoutexchanging the solvent and without precipitating the fluor. However, thegels must be dried prior to film exposure. Water removed by vacuumaspiration contains the water soluble fluor intended for enhancementpurposes, thereby requiring high initial fluor concentration. For thin,porous gels in which mechanical entrapment cannot be relied upon untilsufficient evaporation has occurred to precipitate the water solublefluor, enough fluor can be removed by vacuum aspiration to seriouslydecrease the enhancement process.

Thus, alternative methods for enhancing the detection of radiolabelledmaterials by means of autoradiography or autofluorography are beingsought.

SUMMARY OF THE INVENTION

The present invention features compositions and methods useful inproducing autofluorographs. The compositions of the present inventionhave the added advantage that they are reuseable, with 2-4 uses persolution being standard.

The autoradiography enhancer composition of the invention contains adehydrating agent, an acid anhydride having the formula ##STR1## whereR₁ and R₂ are selected from a group consisting of straight chain, branchchain, and substituted alkyl groups. One to three carbon atoms is thepreferred chain length. The acid anhydride forms 5-95% by weight of thecomposition while 1-47.5% by weight is a water miscible acid, preferablya carboxylic acid. By "water miscible", what is meant is that thechemical is either easily mixed with or soluble with water. Thepreferred acid anhydrides are acetic anhydride, propionic anhydride,trichloroacetic anhydride, and mixtures thereof. The composition alsoincludes a scintillation fluor which forms 0.01-25% by weight. Normally,a low percentage, 0.1-10% of the scintillation fluor is used. Preferredscintillation fluors are selected from a group consisting of 2,5-diphenyloxazole; isopropyl phenyl biphenyloxadiazole;2-[napthyl-(1')]-5-phenyloxazole; t-butylphenyl biphenylyl oxadiazole;p-quatraphenyl acetylene; diphenyl acetylene; 2, 5,diphenyl-3-methyloxazolium salts; 4-chloromethyl-2, 5-diphenyloxazole;polyethylene glycol di-1-naphthylmethyl ether;4[5-(2-phenyloxazolyl)]benzene sulfonic acid; terphenyltrisulfonic acidtrisodium salt; fluorene-2, 7-disulfonic acid disodium salt; 2,5-diphenyl-3-methyloxazolium toluenesulfonate; 4-phthalimido methyl-2,5-diphenyloxazole; 4-aminomethyl-2, 5-diphenyloxazole; and mixturesthereof.

For some uses, it is difficult to have the composition permeatethroughout the separation media. To assist in this and in the laterdrying, a water miscible coupling agent selected from a group consistingof water miscible aliphatic ethers, aliphatic glycol ether esters, andmixtures thereof is used. Preferred coupling agents includediisopropylether, ethoxyethanol acetate, diethylene glycol monoethylether acetate, dipropylene glycol methyl ether acetate, and mixturesthereof. If a coupling agent is used, it normally comprises 1-25% byweight of the composition.

The composition of the present invention may also include ascintillation solvent to assist in capturing the radiation emitted fromthe radioisotope and transferring the energy to the scintillators.Preferred scintillation solvents are 2-methylnaphthalene, naphthalene,and terphenyl compounds. The composition may also include a secondaryfluor. Preferred secondary fluors includep-bis[2-(4-methyl-5-phenyloxazoyl)] benzene; p-bis-(o-methylstyryl)benzene; p-p'-diphenyl stilbene; and 9, 10-diphenyl anthracene.

The invention additionally features a method of autofluorography. Thismethod starts with the step of preparing a separation medium containingradiolabelled materials to be identified. Any standard separationmedium, e.g., chromatographs, electrophoresis gels, or columnchromatography gels can be used. The prepared separation medium issoaked in an excess, preferably a 3-5 fold excess, of an enhancersolution in order to enhance the signals from the incorporatedradiolabelled isotopes. The composition of the invention is thepreferred enhancer solution. The excess enhancer is then decanted andsaved for future use. An aqueous solution may be added to precipitatethe fluor, then the medium is dried. A photographic emulsion, preferablyin the form of a photographic plate, is then exposed by contacting theenhanced separation medium with the plate. The reusability of theenhancer results in significant savings in the cost of the totalautoradiography enhancer fluid and a decreasing amount of radioactiveand hazardous waste produced.

The enhancer may also be used for enhancing autofluorographs on slidesor other sections, e.g., tissue samples or electron micrographs.

The invention is more readily understood by the following detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features an enhancer composition forautofluorography as well as a method of making autofluorographs. Theinvention provides improvements in time and cost efficiency as comparedwith standard autofluorographic processes and compositions.

The invention uses the chemical action of acid anhydrides to act as anin situ dehydrating agent. The combination of the acid anhydride and theacid yields benefits in development and clarity of the image.

The following non-limiting, illustrative examples will further explainthe method and compositions of the invention.

EXAMPLE 1

In this Example, a composition of the invention was tested in fourexperiments, an initial test and three reuses against commerciallyavailable enhancers. The results show that even on reuse, thecomposition of the invention is as good or better than the commerciallyavailable compositions.

An electrophoretogram was prepared using a conventional Laemmli systemtechnique (see Laemmli, Nature (Lond.) 227:680-685 (1970)), yielding a1.5 mm thick 10% polyacrylamide slab gel 12.7 cm by 14 cm. The sampletested was derived from a human kaposi sarcoma cell culture labeled withS³⁵ -methionine for one hour in methionine-free, serum-free media. Theprotein analyzed was Triton® X-100 solubilized protein (solubleproteins), 50,000 cpm analyzed per track (0.03 Ci), about 75% of totalcell proteins.

Electrophoresis was carried out using 10-30 mA current in accordancewith techniques well described in the prior art. After electrophoresis,the drained, water-washed gel was placed in about 4 volumes of asolution having the following compositions by weight: 37.74% aceticanhydride, 47.0% glacial acetic acid, 5.0% diethylene glycol monoethylether acetate, 10.0% 2-methylnaphthalene, 0.25% 2,5 diphenyloxazole and0.01% butylated hydroxytoluene. The gel was gently agitated in a rockerfor 1/2 hour after which the excess liquid was decanted and saved forreuse. About 5-10 volumes of water were added to the gel and gentleagitation resumed for an additional 1/2 hour to precipitate the fluorsystem. The gel was removed from the water and dried with heat andvacuum according to commonly used techniques. The dried gel was then putin contact with Kodak X-AR film for 18 hours at -70° C., and then theexposed film was developed, all according to standard techniques.

This entire procedure was repeated four times additional, the onlyvariation being that the excess decanted liquid which had been saved forreuse, as described above, was serially substituted for the compositiondescribed in the example. Thus a series of five gels were sequentiallytreated using only the original volume of autoradiography enhancerfluid.

There were no noticeable signs of deterioration in the magnitude ofamplification of the enhanced signal until the fifth reuse and eventhen, enhancement was still noticeable. Comparative testing was carriedout using commercially available ENHANCE® and PPO-DMSO, utilizing therespective recommended procedures. The tests showed that the compositionof the invention, on initial use and reuses one-three (four uses total)was equal to or better than ENHANCE® and PPO-DMSO on their respectiveinitial uses. The only problem was that there was some oil or crystalsnoted in the system after the third use.

This Example clearly establishes that the present composition may bereused and still provide excellent enhancement.

EXAMPLE 2

In this Example, a similar composition within the scope of the inventionwas compared to a different commercial fluor system. The compositionused was a solution having the following weight composition wasevaluated: 47.0% glacial acetic acid, 37.72% acetic anhydride, 10.0%2-methylnaphthalene, 5.0% diethylene glycol monoethyl ether acetate,0.25% PPO, and 0.03% butylated hydroxytoluene.

The composition was tested using various samples of ³⁵ S methioninelabelled proteins and with ¹⁴ C standards in gels. The proteins were allimmunoprecipitates.

The enhanced gels using the composition was compared with AUTOFLUOR, acommercially available enhancer, treated gels. The tests showed that thecomposition of the invention was superior to the commercially availableproduct. Again, standard techniques as described in Example 1 were used.

The compositions and methods of the invention yield improvements inenhancement and the ability to handle a wide variety of gels withoutexcess shrinkage. The compositions are reusable, cutting costs and allowsimpler handling techniques than DMSO-based solutions.

Those skilled in the art will determine other embodiments of theinvention.

Such other embodiments are included within the following claims.

What is claimed is:
 1. A method of autofluorography comprising the stepsofa. preparing a separation medium containing radiolabelled materials tobe identified; b. enhancing said prepared separation medium by soakingsaid separation medium in an excess of an enhancer solution, saidenhancer solution being reuseable and comprising1. about 5-95% by weightof an acid anhydride of the formula ##STR2## where R₁ and R₂ areselected from a group consisting of straight chain, branch chain, andsubstituted alkyl groups;
 2. 2. about 0.01-25% by weight of a watermiscible acid; and3. about 0.01-25% by weight of a scintillation fluor;c. decanting said excess of said enhancer solution from said separationmedium wherein said excess is available for reuse; d. drying saidseparation medium; and e. exposing a photographic emulsion to saidenhanced separation medium, thereby forming an autofluorograph.
 2. Themethod of claim 1 further comprising the step of soaking said separationmedium in water to precipitate said fluor component from saidscintillation fluor before said drying step.
 3. The method of claim 1wherein said acid comprises a carboxylic acid.
 4. The method of claim 1wherein said scintillation fluor is selected from the group consistingof 2, 5-diphenyloxazole; isopropyl phenyl biphenyloxadiazole;2-[napthyl-(1')-5-phenyloxazole; t-butylphenyl biphenylyl oxadiazole;p-quatraphenyl acetylene; diphenyl acetylene, and mixtures thereof. 5.The method of claim 1 wherein said scintillation fluor is selected fromthe group consisting of 2, 5, diphenyl-3-methyloxazolium salts;4-chloromethyl-2, 5-diphenyloxazole; polyethylene glycoldi-1-naphthylmethyl ether; 4[5-(2-phenyloxazolyl)] benzene sulfonicacid; terphenyltrisulfonic acid trisodium salt; fluorene-2, 7-disulfonicacid disodium salt; 2, 5-diphenyl-3-methyloxazolium toluenesulfonate;4-phthalimido methyl-2, 5-diphenyloxazole, 4-aminomethyl-2,5-diphenyloxazole; and mixtures thereof.
 6. The method of claim 1wherein said enhancer composition further comprises a water misciblecoupling agent, said coupling agent being selected from the groupconsisting of water miscible aliphatic ethers, aliphatic glycol etheresters, and mixtures thereof.
 7. The method of claim 1 wherein saidenhancer composition further comprises a secondary fluor.
 8. The methodof claim 7 wherein said secondary fluor is selected from the groupconsisting of p-bis[2-(4-methyl-5-phenyloxazoyl)] benzene;p-bis-(o-methylstyryl) benzene; p-p'-diphenyl stilbene; and 9,10-diphenyl anthracene.
 9. The method of claim 1 wherein said enhancercomposition further comprises a scintillation solvent.
 10. The method ofclaim 9 wherein said scintillation solvent is selected from the groupconsisting of 2-methylnaphthalene, naphthalene, and terphenyl compounds.11. The method of claim 1 wherein said acid anhydride is selected fromthe group consisting of acetic anhydride, propionic anhydride,trichloroacetic anhydride, and mixtures thereof.
 12. A method forpreparing autoradiographs of tissue samples or electromicrographscomprising the steps of:a. preparing a tissue sample orelectromicrograph containing radiolabelled materials to be identified;b. enhancing said prepared tissue sample or electromicrograph by soakingor coating said tissue samples or electromicrographs with an excess ofenhancer solution, said enhancer solution being reusable andcomprising1. about 5-95% by weight of an acid anhydride of the formula##STR3## where R₁ and R₂ are selected from a group consisting ofstraight chain, branch chain, and substituted alkyl groups;
 2. about1-47.5% by weight of a water miscible acid; and3. about 0.01-25% byweight of a scintillation fluor; and c. exposing a photographic emulsionto said enhanced tissue samples or electromicrographs, thereby formingan autoradiograph.